Experiments are proposed to: 1) isolate the putative testis determining locus on Yp; 2) understand the mechanism underlying aberrant X/Y interchange, a frequent cause of XX sex reversal; and 3) study whether there is a "spermatogenesis" locus on Yq. 1) Testis determining gene(s) will be isolated by criteria of presence in most XX males and absence in some XY females, evolutionary conservation, and male and/or testis-specific expression. Starting from a deletion map of Yp, a physical map of the testis determining region will be constructed. Y chromosomal DNA sequences derived from large restriction fragments spanning this region will be tested for evolutionary conservation. Their expression will be studied both at the RNA and at the protein level. The gene structure will be analyzed and exons will be sequenced. The ultimate functional proof of candidate testis determining gene(s) will come from their capacity to cause sex reversal in XX transgenic mice. 2) X and Y chromosomal breakpoints will be analyzed in several XX males. This will lead to a further understanding of the underlying mechanism of aberrant X/Y interchange. 3) Using DNA from fertile males with deletions of Yq, a deletion map will be constructed with respect to a putative "spermatogenesis locus". A physical map will allow further delineation of this locus. Expression studies will be performed at the RNA level with evolutionary conserved sequences from this region. The tissue distribution of expressed RNA's will be investigated by in situ hybridization.